Targeting of recombinant Na<sup>+</sup>/glucose cotransporter (SGLT1) to the apical membrane

Kong, Cheng-Te; Varde, Anjali; Lever, Julia E.

doi:10.1016/0014-5793(93)80363-y

Cited by 10 publications

(10 citation statements)

References 29 publications

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“…The identity of the amino acid sequences of the ovine parotid and intestinal proteins directed to the opposite poles of the epithelial cells must indicate that there is no inherent targeting signal present within the protein structure. This conclusion conflicts with that drawn by Kong et al [8]. These authors inserted the entire rabbit SGLT1 cDNA into a mammalian expression vector and demonstrated that when this vector was used to transfect MDCK cells, SGLT1 activity was expressed.…”

Section: Immunodetection Of Sglt1-type Proteinsmentioning

confidence: 77%

“…In rabbits, the kidney and intestinal apically located SGLT1 proteins appear to arise from a single gene [7]. It has been concluded that the information for targeting the rabbit intestinal SGLT1 protein to the apical membrane is contained in the primary amino acid sequence of the SGLT1 protein [8]. The location of the Na+-dependent D-glucose symporter on the basal membrane of the secretory acinar cells of the parotid gland presents an opportunity to investigate the mechanism(s) whereby the apically and the basally located proteins are targeted to these two different domains of the plasma membrane.…”

Section: Introductionmentioning

confidence: 99%

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Amino acid sequence and the cellular location of the Na+-dependent d-glucose symporters (SGLT1) in the ovine enterocyte and the parotid acinar cell

Tarpey

Wood

Shirazi-Beechey

et al. 1995

Biochemical Journal

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The Na(+)-dependent D-glucose symporter has been shown to be located on the basolateral domain of the plasma membrane of ovine parotid acinar cells. This is in contrast to the apical location of this transporter in the ovine enterocyte. The amino acid sequences of these two proteins have been determined. They are identical. The results indicated that the signals responsible for the differential targeting of these two proteins to the apical and the basal domains of the plasma membrane are not contained within the primary amino acid sequence.

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Section: Immunodetection Of Sglt1-type Proteinsmentioning

confidence: 77%

Section: Introductionmentioning

confidence: 99%

Amino acid sequence and the cellular location of the Na+-dependent d-glucose symporters (SGLT1) in the ovine enterocyte and the parotid acinar cell

Tarpey

Wood

Shirazi-Beechey

et al. 1995

Biochemical Journal

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“…Cells were grown in 10 cm dishes and prepared as reported [19]. In brief, cells were washed with PBS once, then solubilized in lysis buffer (50 mM Tris-HCl, pH 8, 150 mM NaCl, 0.02% sodium azide, 100 g/ml PMSF, 1 g/ml aprotinin, 1% Triton X) and harvested.…”

Section: Western Immunoblottingmentioning

confidence: 99%

“…The separated proteins were transferred to Immobilon membranes (Millipore, Eschborn, Germany) using an electroblotting system (BioRad, München, Germany). The filters were blocked for two hours at room temperature with 5% dry milk and 0.05% Tween 20 (Flika, Buchs, Switzerland) in Tris-buffered saline and incubated with antibodies specific for GLUT1 and GLUT3 [19] (East Acres, Wak Chemie, Bad Homburg, Germany) at a concentration of 1:800 and 1:500 respectivley. Visualization of bound antibodies was carried out with the ECL system (Amersham).…”

Section: Western Immunoblottingmentioning

confidence: 99%

Expression of glucose transporters in human peritoneal mesothelial cells

Schröppel

Fischereder

Wiese

et al. 1998

Kidney International

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Glucose containing solutions, the basis of peritoneal dialysis fluids, affect the proliferation and regeneration of peritoneal mesothelial cells (MsC). The aim of this study was to examine mechanisms of glucose transport into MsC, that is, the expression of facilitative glucose transporters (GLUT) and the Na(+)-dependent glucose transporter (SGLT1) in human primary MsC and a transfected MsC line. Since expression of both transporters is differentiation dependent, we investigated the effects of cell differentiation induced by culturing MsC on membranes or by addition of hexamethylene bisacetamide (HMBA; 6 mM), which enhances SGLT1 expression in LLC-PK1 cells. Levels of mRNA for GLUT1 through GLUT4 and SGLT1 were evaluated by reverse transcriptase-polymerase chain reaction (RT-PCR). The presence of the corresponding proteins was examined by Western blotting and localized by immunofluorescence. Active, Na(+)-dependent glucose transport was assessed by alpha-methyl-D-[14C]glucopyranoside (AMG) with and without the SGLT1-specific inhibitor phlorizin and by patch clamp experiments in NaCl or choline-chloride, For Na(+) dependent glucose uptake choline chloride instead of NaCl served as negative control. Facilitative transport was assessed using 2-fluoro-2-deoxy-[14C]-D-glucose (FDG) with and without the inhibitors cytochalasin B or phloretin. Primary and transfected MsC express GLUT1 and GLUT3 mRNA while no transcripts were found for GLUT2 and GLUT4. No SGLT1 transcript was detectable in subconfluent cells. Semiquantitative RT-PCR analysis documented that the addition of the differentiation inducer HMBA to confluent cultures or growth of MsC on membranes for seven days produced a down-regulation of mRNA for GLUT1, no change for GLUT3, and a substantial increase for SGLT1 mRNA. Under these conditions MsC express SGLT1 protein and possess a Na(+)-dependent glucose uptake as assessed by AMG. Phlorizin (1 mM) inhibits AMG uptake by 30 to 40%. In patch clamp experiments the addition of extracellular glucose depolarized the membrane potential only in the presence of sodium. These results indicate that differentiated MsC express GLUT1, GLUT3, and SGLT1. Further characterization of these transport mechanisms and their regulation may help to understand the cellular effects of glucose on MsC in peritoneal dialysis.

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“…Which parts of the proteins are involved in Na ϩ and sugar binding? To begin to answer these important questions, we generated a chimeric DNA-construct corresponding to amino acids 1-380, which form the putative transmembrane helices 1-8 of pSGLT2, and amino acids 381-662 (which form putative helices 9 -14) of pSGLT1 (19) (20). The product of this three-fragment ligation was a chimeric sequence, coding for amino acids 1-380 from pig SGLT2 and amino acids 381-662 from pig SGLT1 (2.4-kilobase BamHI/XhoI coding fragment).…”

mentioning

confidence: 99%

Sugar Binding to Na+/Glucose Cotransporters Is Determined by the Carboxyl-terminal Half of the Protein

Panayotova‐Heiermann¹,

Loo²,

Kong³

et al. 1996

Journal of Biological Chemistry

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D-Glucose is absorbed across the proximal tubule of the kidney by two Na ؉ /glucose cotransporters (SGLT1 and SGLT2). The low affinity SGLT2 is expressed in the S1 and S2 segments, has a Na ؉ :glucose coupling ratio of 1, a K 0.5 for sugar of ϳ2 mM, and a K 0.5 for Na ؉ of ϳ1 mM. The high affinity SGLT1, found in the S3 segment, has a coupling ratio of 2, and K 0.5 for sugar and Na ؉ of ϳ0.2 and 5 mM, respectively. We have constructed a chimeric protein consisting of amino acids 1-380 of porcine SGLT2 and amino acids 381-662 of porcine SGLT1. The chimera was expressed in Xenopus oocytes, and steadystate kinetics were characterized by a two-electrode voltage-clamp. The K 0.5 for ␣-methyl-D-glucopyranoside (0.2 mM) was similar to that for SGLT1, and like SGLT1 the chimera transported D-galactose and 3-O-methylglucose. In contrast, SGLT2 transports poorly D-galactose and excludes 3-O-methylglucose. The apparent K 0.5 Na was 3.5 mM (at ؊150 mV), and the Hill coefficient ranged between 0.8 and 1.5. We conclude that recognition/transport of organic substrate is mediated by interactions distal to amino acid 380, while cation binding is determined by interactions arising from the amino-and carboxyl-terminal halves of the transporters. Surprisingly, the chimera transported ␣-phenyl derivatives of D-glucose as well as the inhibitors of sugar transport: phlorizin, deoxyphlorizin, and ␤-D-glucopyranosylphenyl isothiocyanate are transported with high affinity (K 0.5 for phlorizin was 5 M). Thus, the pocket for organic substrate binding is increased from 10 ؋ 5 ؋ 5 (Å) for SGLT1 to 11 ؋ 18 ؋ 5 (Å) for the chimera.Reabsorption of D-glucose from the glomerular filtrate along the proximal tubules of the kidney occurs by two apparently distinct cotransport systems (1-3). In the S1 and S2 segments of the renal convoluted tubule, reabsorption is mediated to 90% by a low affinity Na ϩ /glucose cotransporter (SGLT2) 1 with an apparent K 0.5 for sugar of 6 mM and a Na ϩ :glucose coupling ratio of 1. The reabsorption process is completed in the S3 segment by another Na ϩ /glucose cotransporter (SGLT1) with significantly higher apparent affinity for sugar (K 0.5 ϳ0.35 mM) than SGLT2 and a Na ϩ /glucose coupling ratio of 2.The pig renal cell line LLC-PK1 (4) has often been used as a model system to study glucose transport in kidney proximal epithelia. LLC-PK1 cells express Na ϩ /glucose transport activity (apparent K 0.5 D-GLC ϳ0.28 mM; Refs. 5 and 6) located at the apical surface (7). The presence of SGLT1 (2:1 Na ϩ /glucose stoichiometry) in these cells was shown by Misfeldt and Sanders (8) and Moran et al. (9). In 1990, Ohta et al. (10) isolated a SGLT1 cDNA clone (pSGLT1) from LLC-PK1 cells, with 84% and 87% identity to the high affinity rabbit and human SGLT1. There are three electrophysiologically characterized isoforms of the high affinity Na ϩ -dependent glucose transporter, the rat, human, and rabbit SGLT1s (11-16). All three isoforms show an apparent K 0.5 ␣MDG between 0.17 and 0.49 mM, an apparent K 0.5 Na of 2-7 mM, and a Hil...

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Targeting of recombinant Na⁺/glucose cotransporter (SGLT1) to the apical membrane

Cited by 10 publications

References 29 publications

Amino acid sequence and the cellular location of the Na+-dependent d-glucose symporters (SGLT1) in the ovine enterocyte and the parotid acinar cell

Amino acid sequence and the cellular location of the Na+-dependent d-glucose symporters (SGLT1) in the ovine enterocyte and the parotid acinar cell

Expression of glucose transporters in human peritoneal mesothelial cells

Sugar Binding to Na+/Glucose Cotransporters Is Determined by the Carboxyl-terminal Half of the Protein

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Targeting of recombinant Na+/glucose cotransporter (SGLT1) to the apical membrane

Cited by 10 publications

References 29 publications

Amino acid sequence and the cellular location of the Na+-dependent d-glucose symporters (SGLT1) in the ovine enterocyte and the parotid acinar cell

Amino acid sequence and the cellular location of the Na+-dependent d-glucose symporters (SGLT1) in the ovine enterocyte and the parotid acinar cell

Expression of glucose transporters in human peritoneal mesothelial cells

Sugar Binding to Na+/Glucose Cotransporters Is Determined by the Carboxyl-terminal Half of the Protein

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Targeting of recombinant Na⁺/glucose cotransporter (SGLT1) to the apical membrane